Stressful stimuli, including growth factor deprivation, irradiation, and chemotherapeutic drugs, induce cell death via apoptosis. In most cases, these stimuli initiate pathways that culminate in mitochondrial outer membrane permeabilization and release of cytochrome c from the intermembrane space into the cytoplasm. Cytochrome c then induces (d)ATP-dependent oligomerization of apoptosis protease-activating factor-1 (Apaf-1) into a multimeric apoptosome complex that sequentially recruits and activates the initiator caspase-9 and the effector caspase-3. Despite advances in our understanding of this critically important complex, fundamental questions remain unanswered. Indeed, significant controversy surrounds the composition and size of the apoptosome, and it remains unclear whether caspase-9 undergoes activation within the apoptosome in response to dimerization and/or Apaf-1-induced conformational changes. In aim #1, we will utilize sophisticated techniques, including analytical ultracentrifugation, site-specific incorporation of an unnatural/cross-linkable amino acid, and synchrotron protein footprinting assays, among others, to determine the stoichiometric, dimerization, and conformational status of caspase-9 within the Apaf-1 apoptosome complex. Similarly, a number of previous studies suggest that phosphorylation of procaspase-9 at various sites by a number of kinases inhibits or activates this protease. However, a number of these studies are controversial, and it remains entirely unknown, mechanistically, how phosphorylation alters the activation/activity of procaspase-9. In aim #2, we will determine if phosphorylation of procaspase-9 impacts its affinity for the apoptosome, or its ability to dimerize or undergo conformational changes necessary for activation. The impact of phosphorylation on apoptosis will also be assessed, in part, through reintroduction of phosphomutants into caspase-9-deficient cell lines. Finally, we have recently demonstrated that the Apaf-17caspase-9 apoptosome functions as a proteolytic-based molecular timer, wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, pro-caspase-9 autoprocessing activates the timer, and the rate at which processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer ticks over. In aim #3, we will assess the importance of this molecular timer in vivo using a novel caspase-9 knock-in mouse that prevents procaspase-9 from undergoing processing. We will determine if this disengagement of the timer sensitizes animals to developmental or toxicant-induced apoptosis. In summary, the major goal of this grant application is to utilize a number of highly novel approaches, never before brought to bear on the Apaf-17caspase-9 apoptosome complex, in order to characterize in molecular detail the mechanisms that mediate the activation and regulation of this critical caspase-activating complex. Moreover, these studies will improve our general understanding of how initiator caspases are activated within large protein complexes and will shed light on how they can be exploited therapeutically in the future to treat diseases ranging from cancer to neurodegeneration.